Identification of Functional Platelet-Activating Factor Receptors on Human Keratinocytes

Abstract

Platelet-activating factor (PAF) is a potent inflammatory mediator that has been shown to be produced by human keratinocytes and is thought to play a role in cutaneous inflammation. Immunofluorescence and radioligand binding studies were used to characterize PAF receptors (PAF-R) on human keratinocytes and the human epidermoid cell lines A-431 and HaCaT. Indirect immunofluorescence studies demonstrated anti-PAF-R staining of primary cultures of human keratinocytes, A-431 cells, and HaCaT cells. Primary cultures of human fibroblasts and the melanoma cell line SK-30 failed to show immunostaining above that seen with control antiserum. With indirect immunofluorescence studies of sections of normal human skin, a granular anti-PAF-R staining pattern was noted on the keratinocyte cell membranes. A-431 cells readily metabolized PAF by deacetylation-reacylation at 37 degrees C, but not at 4 degrees C. Binding studies on crude membrane preparations of A-431 cells conducted at 4 degrees C demonstrated specific binding that reached saturation by 120 min. Scatchard analysis of PAF binding data revealed a single class of high-affinity (KD = 6.3 +/- 0.3 nM) PAF binding sites. The immunofluorescence and radioligand binding sites were shown to be functional PAF-Rs, as 10 pM to 1 microM PAF increased intracellular calcium in primary cultures of human keratinocytes, A-431 cells, and HaCaT cells, whereas PAF treatment of primary cultures of human fibroblasts or the melanoma cell line SK-30 did not result in changes in the intracellular calcium concentration. The structurally dissimilar PAF-R antagonists CV-6209, Ro19-3704, and alprazolam all inhibited the PAF-induced calcium changes in A-431 cells. The CV-6209 inhibition was seen at doses that competed with the PAF binding to these cells. These studies provide the first evidence for the presence of a functional PAF-R expressed on human keratinocytes, suggesting that this lipid mediator may play an important role in normal keratinocytes or in inflammatory dermatology.

Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor

“…Intracellular Ca 2+ mobilization studies in KB cells were conducted using Fura-2 AM (Invitrogen Corp.) as previously described (22).…”

“…of biologically active metabolites), our laboratory previously created a cellular model system by transduction of the PAF-R into a PAF-R-deficient epidermal cell line to study the role of the PAF-R in epithelial cell biology. Unlike normal human keratinocytes and the human keratinocyte-derived carcinoma cell line HaCaT (22), the human epidermal carcinoma cell line KB does not express functional PAF-R. A PAF-R-positive KB cell line, KBP, was created by transducing KB cells with a replication-deficient MSCV2.1 retrovirus containing the human PAF-R cDNA. KB cells were also transduced with the retroviral empty vector alone to establish a transduction control cell line, KBM.…”

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

“…The PAF receptor is expressed by cells of the innate immune system, but also by keratinocytes of the skin 14 . PAF is the most potent phospholipid agonist yet identified where high affinity recognition depends on its sn-1 ether bond and a short sn-2 acetyl residue.…”

PAF, a Putative Mediator of Oral Inflammation

“…have been identified in keratinocytes in human and rat skin as well as in cultured human epidermoid cell lines (Fisher et al, 1989;Travers et al, 1995;Shimada et al, 1998). Moreover, both acyl-linked and alkyl-linked acetylated phospholipids have been identified in stimulated human keratinocytes in culture (Travers et al, 1996(Travers et al, , 1997 (1) PAF has direct and potent effects upon cardiac function and has been implicated in cardiovascular (patho)physiology (Evangelou, 1994;Prescott et al, 1996Prescott et al, ,1997Feuerstein et al, 1997;Yamada et al, 1998); (2)PAF-like phospholipids (oxidized-PC) and PAF-AH may be involved in the pathogenesis of atherosclerosis (see above); (3)salivary PAF levels are significantly increased in subjects with periodontal disease (Fig.…”